Ischemia/reperfusion (I/R) injury is an important pathologic problem in liver resection and liver transplantation. Studies in animal models have implicated liver macrophages (Kupffer cells) and neutrophils in the pathogenesis of I/R liver injury wherein Kupffer cell-derived reactive oxygen species (ROS) in hepatic sinusoids contribute to vascular and parenchymal cell injury and the recruitment of neutrophils into the liver. Due to the complexity of in vivo models, the role of ROS in signaling and the cellular and molecular mechanisms of reperfusion-induced hepatic endothelial injury remain unresolved, as are the specific contribution of Kupffer cells and hepatocytes to this injury process. The current project proposes to (a) establish in vitro models of sinusoidal endothelial cell monolayers that are cocultured with either Kupffer cells and/or hepatocytes to simulate the cellular hierarchy in vivo, and (b) employ these models of cellular complexity to delineate the molecular basis for reoxygenation-induced hepatic vascular injury. Our central working hypothesis is that anoxia/reoxygenation (A/R) induces an imbalance in sinusoidal endothelial ROS and nitric oxide (NO) that leads to activation of NFkB and enhanced neutrophil-endothelial cell interaction. We further hypothesize that this inflammatory response is potentiated by mediators derived from Kupffer cells and hepatocytes. The aims address 3 specific hypothesis. Aim 1. To test the hypothesis that A/R induces sinusoidal endothelial ROS/NO imbalance that leads to activation of NFkB, expression of inflammatory cytokines, upregulation of adhesion molecules and enhanced neutrophil-endothelial cell interactions. Aim 2. To test the hypothesis that inflammatory mediators derived from Kupffer cells potentiate A/R-induced sinusoidal endothelial dysfunction during early reoxygenation. Aim 3. To test the hypothesis that hepatocyte-derived mediators contribute to late phase A/R-induced sinusoidal endothelial cell inflammatory response and vascular dysfunction. The results will provide important insights into the mechanism of vascular I/R injury in the liver and will identify potential targets for therapeutic intervention.